A Fos-Jun element in the first intron of an α2u-globulin gene

The hepatic expression of the _2u gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect -globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an _2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M₂ peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG₃ hepatoma cells treated with TPA. Co-transfection withfos andjun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.     

Primary structure of elongation factor 1β from Artemia

cDNA complementary to mRNA coding for the elongation factor EF-1β has been cloned. A γgt 11 cDNA library has been screened with an antiserum against EF-1β which exchanges GDP bound to EF-1α with exogenous GTP during protein synthesis. The derived amino acid sequence corresponds to 208 amino acids including the N-terminal methionine which is absent in the mature protein. About sixty percent of the protein was sequenced by direct protein sequence analysis. Comparison of Artemis salina EF-1β with Escherichia coli EF-Ts shows no evident homology.     

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

The complete intron/exon structure of Ephydatia mülleri fibrillar collagen gene suggests a mechanism for the evolution of an ancestral gene module

We have completed the analysis of a genomic clone, G238, that contains most of the coding region of the sponge COLF1 fibrillar collagen gene. The main triple helical domain is encoded by 31 exons. Except for the 5 junction exon and the two last 3 exons (126 and 18 base pairs), all these exons are related to a 54-bp unit and begin with an intact glycine codon. A good correlation can be made between this sponge gene and a vertebrate fibrillar collagen gene, revealing the high conservation of the members of this family during evolution. The reconstitution of an ancestral collagen gene can be made by considering all the exon/intron junctions of these genes. We suggest that such an ancestral gene arose from multiple duplications of a 54-bp exon and a (54 + 45)-bp module.Abbreviations used bp base pair(s) - kb kilobase(s) - C-protease the enzyme that cleaves the carboxyl-terminal propeptide     

Molecular cloning,characterization and expression of an elongation factor 1α gene in maize

A cDNA (zmEF1A) and the corresponding genomic clone (zmgEF1A) of a member of the gene family encoding the subunit of translation elongation factor 1 (EF-1) have been isolated from maize. The deduced amino acid sequence is 447 residues long interrupted by one intron. Southern blot analysis reveals that the cloned EF-1 gene is one member out of a family consisting of at least six genes. As shown by northern hybridizations in leaves the mRNA level increases at low temperature whereas time-course experiments over 24 h at 5°C show that in roots the overall mRNA level of EF-1 is transiently decreased. These results indicate that the expression of EF-1 is differently regulated in leaves and roots under cold stress.     

A transducing bacteriophage λ carrying the structural gene for elongation factor Ts

Summary A specialized transducing bacteriophage dpolCdapD-9 has been isolated that carries the structural gene for EF-Ts¹ (tsf). The presence of EF-Ts among the proteins synthesized under the direction of this phage in UVL-inactivated cells has been detected by two-dimensional gel electrophoresis and has been verified by antibody precipitation. In an induced lysogen of this phage the relative rate of synthesis of EF-Ts is increased 4-fold. Evidence is presented which suggests that the structural genes for ribosomal protein S2 (rpsB) and RNA polymerase factor (sit) also lies on dpolCdapD-9.     

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1α is conserved among angiosperms

In Arabidopsis thaliana, the activation process of the A1 EF-1 gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5 non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5 intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.     

Rapid fold and structure determination of the archaeal translation elongation factor 1β from Methanobacterium thermoautotrophicum

The tertiary fold of the elongation factor, aEF-1, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1 was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1 structure revealed close similarity to its human analogue, eEF-1. In agreement with studies on EF-Ts and human EF-1, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1. aEF-1 was also found to bind calcium in the groove between helix 2 and strand 4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.     

Identification of the first deletion–insertion involving the complete structure of GAA gene and part of CCDC40 gene mediated by an Alu element

Pompe disease is an uncommon autosomal recessive glycogen storage disorder caused by deficiency of acid α-glucosidase. Classic infantile form triggers severe cardiomyopathy, hypotonia, and respiratory failure, leading to death within the first two years of life. The majority of patients with Pompe disease have been reported to have point mutations in the GAA gene. We report the first complex deletion–insertion encompassing the complete structure of GAA gene and a large fragment of the gene CCDC40 in a patient with very severe form of Pompe disease. Sequencing analysis of breakpoints allowed us to determine the potential implication of an Alu repeat in the pathogenic mechanism. We suggest that molecular strategy of Pompe disease should include systematic analysis of large rearrangements.     

The initiation site for recombination cog is at the 3′ end of the his-3 gene in Neurospora crassa

Summary The response of Nicotiana tabacum to tentoxin (chlorosis) is inherited with chloroplasts. N. tabacum var. Xanthi, a tentoxin-resistant line, was used to pollinate tentoxin-sensitive N. tabacum line 92, an alloplasmic male-sterile line containing N. undulata plastids. The seeds were mutagenized with nitrosomethylurea and germinated in the presence of tentoxin. Two percent of the seedlings had green sectors in their first true leaves. These plants were grown to maturity under non-selective conditions. Homogeneous tentoxin-resistant lines were obtained in the third generation. DNA analysis indicated, however, that selection for paternal plastids, rather than mutagenesis of maternal ones, had occurred in the tentoxin-resistant progeny. Mitochondria, which were not under selection pressure, were inherited maternally as expected. Inheritance of tentoxin-resistant paternal plastids did not require seed mutagenesis. Normally germinated seedlings that were kept under tentoxin selection consistently produced a low level of resistant green sectors in their first true leaves. Thus, normal, low-frequency transmission of paternal plastids in N. tabacum can be directly revealed by using tentoxin.     

Excision of a Ds-like maize transposable element (AcΔ) in a transient assay in Petunia is enhanced by a truncated coding region of the transposable element Ac

Summary The excision of a Ds-like transposable element (Ac) is mediated in trans by the transposable element Ac or its derivatives in Petunia protoplasts cotransfected with two plasmid DNAs. Excision restores the activity of the -glucuronidase (GUS) gene that is otherwise shut off by the presence of Ac in its leader sequence. A transient expression assay (histochemical test) is used to detect the -glucuronidase activity at the protoplast level. The number of blue-stained protoplasts is a measure of the excision frequency. With Ac alone a near-zero background of GUS activity is detected, which is weakly enhanced by the presence, in trans, of either the wild-type Ac or the coding region (ORF_a) transcribed from the 2 promoter of Agrobacterium tumefaciens TR-DNA. A strong enhancement is observed when a truncated Ac coding region, also under the control of the 2 promoter, is supplied in trans. The truncated version has ATG₁₀ at codon 103 in frame with ORF_a and is preceded by 7 out-of-frame ATGs. The assay is quick and well suited for detection of excision frequencies above the value obtained with the wild-type Ac. The presence of empty donor sites following excision can be demonstrated by PCR amplification and direct sequencing of the appropriate DNA fragment.     

Overexpression of Arabidopsis thaliana Na+/H+ antiporter gene enhanced salt resistance in transgenic poplar (Populus?×?euramericana ‘Neva’)

Salinity is a major abiotic stress factor limiting plant growth and productivity. One possible method to enhance plant salt-resistance is to compartmentalize sodium ions away from the cytosol. In the present work, a vacuolar Na⁺/H⁺ antiporter gene AtNHX1 from Arabidopsis thaliana, was transferred into Populus × euramericana ‘Neva’ by Agrobacterium tumefaciens in order to enhance poplar salt-resistance. The results showed that the transgenic poplar were more resistant to NaCl than the wild-type (WT) in greenhouse condition. Compared with the WT, plant growth and photosynthetic capacity of the transgenic plants were enhanced, and the transgenic plants accumulated more Na⁺ and K⁺ in roots and leaves under the same NaCl condition, whereas malondialdehyde and relative electrical conductivity were lower. All of these properties of the transgenic poplar were likely to be a consequence of the overexpression of AtNHX1 caused Na⁺ sequestration in the vacuoles and improved K⁺ absorption, thus reducing their toxic effects. These results indicated overexpression of the AtNHX1 enhanced salt-resistance of poplar, and AtNHX1 played an important role in the compartmentation of Na⁺ into the vacuoles. Therefore, this study provides an effective way for improving salt resistance in trees.